Impact of habitual chewing on gut motility via microbiota transition

The gut environment, including the microbiota and its metabolites and short-chain fatty acids (SCFA), is essential for health maintenance. It is considered that functional recovery treatment for masticatory dysphagia affects the composition of the gut microbiota, indicating that habitual mastication, depending on the hardness of the food, may affect the gut microbiota and environment. However, the impact of chronic powdered diet feeding on the colonic condition and motility remains unclear. Here, we evaluated various colonic features in mice fed with powdered diets for a long-term and a mouse model with masticatory behavior. We observed a decreased abundance of the SCFA-producing bacterial genera in the ceca of the powdered diet-fed mice. Based on the importance of SCFAs in gut immune homeostasis and motility, interestingly, powdered diet feeding also resulted in constipation-like symptoms due to mild colitis, which were ameliorated by the administration of a neutrophil-depleting agent and neutrophil elastase inhibitors. Lastly, the suppressed colonic motility in the powdered diet-fed mice was significantly improved by loading masticatory activity for 2 h. Thus, feeding habits with appropriate masticatory activity and stimulation may play a key role in providing a favorable gut environment based on interactions between the gut microbiota and host immune system.

. Antibodies used in this study.

Supplementary Figure S1 Information
The following experiments were performed to investigate the influence of long-term powdered diet-feeding on the levels of acetate, propionate, and n-butyrate in the plasma. These methods have been explained in the text. There were no significant changes in SCFA levels in the plasma between the powdered diet-and control diet-fed groups (Supplementary Figure S1). Figure S1. Effects of long-term powdered diet feeding on short-chain fatty acid (SCFA) levels in the plasma of mice fasted for 5 h. The levels of acetate, propionate, and nbutyrate among SCFAs extracted from the samples collected from the plasma (plasma; p = 0.6025, 0.2936, and 0.6785, respectively, n = 8/group). Student's t-test. Data are presented as mean ± SEM for each group.

Supplementary Figure S2 Information
The following experiments were performed to investigate the effect of sodium butyrate on constipation-like symptoms in the powdered diet-fed mice. Immediately after intraperitoneal administration of sodium butyrate (1200 mg/kg, i.p.) or saline to non-fasted mice, they were individually placed in plastic chambers. Two hours later, their feces were counted and collected.
The fecal moisture content was calculated as the difference between wet and dry weights of the feces. Administration of butyrate significantly reduced the number of fecal pellets, but not total fecal weight and fecal moisture content, in the long-term powdered diet-fed mice (Supplementary Figure 2). Figure S2. Effect of sodium butyrate (1200 mg/kg, i.p.) on the number of fecal pellets, total fecal weight, and fecal water content in the long-term powdered diet-fed mice (p = 0.0176, 0.5569, and 0.9159, respectively, n = 5-10/group). Student's t-test. Data are presented as mean ± SEM for each group. *p < 0.05; saline-treated powdered diet-fed mice group.

Supplementary Figure S3. Information
The following experiments were performed to investigate the effect of sivelestat on the number of fecal pellets, total fecal weight, and fecal moisture content in the control diet-fed mice. These methods have been explained in the text. There were no significant differences in the number of 4 fecal pellets, total fecal weight, and fecal moisture content in the control diet-fed group (Supplementary Figure S3). Figure S3. Effect of sivelestat (50 mg/kg, i.p.) on the number of fecal pellets, total fecal weight, and fecal water content in the control diet-fed mice (p = 0.0567, 0.4932, and 0.8418, respectively, n = 7-8 group). Student's t-test. Data are presented as mean ± SEM for each group.

Uncropped western blot images
The band images at the bottom of each figure in the main text were cropped from these western blot images. LAS 4010 (GE Healthcare) was used to visualize them. In Figures S4 to S7, the lanes indicated with the thick arrows were used for the cropped images in each of the figures.
Furthermore, the lanes indicated with the thin arrows are related to quantification. Figure S4.
However, the samples derive from the same experiment and the experiments were processed in parallel.